Pseudomyxoma peritonei

* Context.-Carcinomas of the appendix are usually well-- differentiated mucinous adenocarcinomas that tend to produce pseudomyxoma peritonei and do not show metastatic spread until late in the disease process. In contrast, adenocarcinomas of the colon and rectum rarely result in pseudomyxoma peritonei and frequently metastasize, even if mucinous and well differentiated. These differences in behavior may be reflected by differences at the molecular level.

Objectives.-To examine adenocarcinomas and their precursor lesions (adenomas) of the appendix and colorectum and to determine whether differences exist in the numbers of proliferating and apoptotic cells or in expression of p53, bcl-2, and the standard form of CD44 (CD44s).

Design.-Retrospective analysis of surgical specimens.

Setting.-Multicenter study.

Patients.-Individuals treated surgically for tumors of the appendix or colorectum.

Interventions.-Sections were cut from formalin-fixed surgical specimens and immunohistochemical tests were

performed for Ki-67 (as a marker of proliferating cells), M30 (as a marker of apoptotic cells), p53, CD44s, and bcl-2.

Main Outcome Measures.-Expression of Ki-67, M30, p53, CD44s, and bcl-2 in tumor cells.

Results.-The appendiceal adenomas showed significantly lower Ki-67 counts, p53 expression, and bcl-2 expression. When compared with adenocarcinomas of the colorectum in general (mucinous and nonmucinous), the appendiceal adenocarcinomas showed significantly lower Ki-67 counts, M30 counts, and CD44s expression. However, when the analysis was confined to well-differentiated mucinous adenocarcinomas, only the M30 count was significantly different.

Conclusions.-The lower proliferative and apoptotic activity of appendiceal carcinomas and the lower CD44s expression are in keeping with their more indolent behavior compared with adenocarcinomas of the colorectum. However, when only the subset of well-differentiated mucinous adenocarcinomas was compared, only the apoptotic activity was different, suggesting that the other differences were related to the morphologic structure of the lesions.

(Arch Pathol Lab Med. 2002;126:837-841)

Adenocarcinomas of the appendix are usually characterized by abundant mucin production, well-differentiated morphologic structure, relatively indolent behavior, and a predilection for producing the clinical picture of pseudomyxoma peritonei, in which carcinoma spreads along peritoneal surfaces with little penetration of the underlying organs.1-4 In contrast, adenocarcinomas of the colon, even when mucinous, rarely produce pseudomyxoma peritonei and typically spread by lymphatic and hematogenous metastasis. Differences between the appendix and the colorectum are also seen in the precursors of adenocarcinoma. Adenomas with a purely villous architecture or serrated pattern together with well-differentiated, mucin-rich epithelium form a large proportion of appendiceal adenomas, whereas such lesions are relatively unusual in the rest of the large intestine.1,2

It is possible that the differences in morphologic structure and behavior in adenomas and carcinomas of the appendix and colorectum may be associated with differences in cell proliferation, apoptosis, and expression of related molecules. Both p53 and bcl-2 are believed to be important in oncogenesis of many neoplasms. p53 is a nuclear protein that plays a central role in cell cycle regulation; it induces cell cycle arrest or apoptosis in response to DNA damage, and mutations are frequently associated with colorectal oncogenesis.5-7 Loss of wild-type p53 expression due to mutations of TP53 is frequently associated with accumulation of abnormal p53 within cells due to increased protein stability.8,9 The detection of p53 immunohistochemically has been used as a surrogate marker for TP53 mutation. However, caution is needed in the interpretation of p53 immunostains, since wild-type p53 can be detected with appropriate antigen retrieval techniques, especially where DNA damage is occurring, and some mutations of TP53 result in absence of p53 protein. Small numbers of scattered cells are more likely to represent wild-type expression than mutant p53 in colorectal carcinomas.8,9

The product of the proto-oncogene bcl-2 inhibits programmed cell death10 and may be down-regulated by p53.6 Colonic adenocarcinomas tend to show less bcl-2 expression than adenomas.11-15

CD44 is a cell surface glycoprotein found in many different cells that binds hyaluronate, laminin, collagen, and fibronectin.16,17 Various forms of CD44 are produced by alternative splicing of the messenger RNA. The hemopoietic or standard form of CD44 (CD44s) is widely distributed in healthy and neoplastic tissues,16,17 including adenomas and carcinomas of the large intestine.18 Loss of expression has been associated with an increased risk of lymph node metastases.19 If CD44s expression is reintroduced by transfection into human cell lines that lack it, the cells exhibit reduced growth rate and tumorigenicity.20,21 However, increased expression of CD44s in carcinomas of the large intestine has been associated with an increased risk of liver metastases.22

This study was designed to test the hypothesis that adenomas and adenocarcinomas of the colorectum differ from those of the appendix in apoptotic count, cell proliferation, and immunoreactivity for bcl-2, p53, and CD44. Since more carcinomas of the appendix are mucinous and well differentiated than in the colorectum,2 any differences between the 2 sites might simply be due to this factor. Therefore, we decided that we would reexamine any data that showed significant differences between the 2 sites, confining the analysis to well-differentiated mucinous carcinomas.

MATERIALS AND METHODS

Cases were retrieved from the archives of 3 institutions: St Mark's Hospital, London, England, the Royal Air Force Institute of Pathology and Tropical Medicine, Halton, England, and the Armed Forces Institute of Pathology, Washington, DC. Only cases with formalin-fixed, paraffin-embedded tissue available were included. For morphologic assessment, 5-wm-thick sections were stained with hematoxylin-eosin. The study was performed in accordance with the provisions of the Armed Forces Institute of Pathology Human Use Committee and the statutes of the US Privacy Act.

Ki-67 (clone Ki-67; Dako, Glostrup, Denmark) was used as a marker of proliferating cells; compared with other immunohistochemical measures of proliferation, it is more specific for proliferative activity and results do not depend so heavily on variations in fixation.23,24 We used clone DF1485 (Dako) for detection of CD44s. This antibody recognizes a trypsin-resistant epitope of the CD44 molecule, and its pattern of immunoreactivity is compatible with CD44s.19,25 M30 (Roche Diagnostics, Lewes, Sussex, England) is a monoclonal antibody that recognizes a neoepitope of cytokeratin 18 produced by caspase activity during apoptosis. Results of M30 and in situ end labeling correlate strongly, and M30 has some technical advantages over in situ end labeling.26 The expression of p53 was demonstrated with clone DO-7 (Dako)27,28; bcl-2 was demonstrated with clone 124 (Dako).27,29

The immunohistochemical reactions were performed using the avidin-biotin method following standard procedures. Tissue sections were cut at 5 (mu)m, dewaxed, and hydrated in xylene and ethanol before antigen retrieval. For Ki-67, p53, bcl-2, and CD44s, antigen retrieval was performed by placing the slides in a citrate buffer (0.01M citric acid with 80 g/L of sodium hydroxide, adjusted to pH 6.0) and heating in a microwave (Energy Beam Sciences model H2800; The Laboratory Microwave Company, Agawam, Mass) at 95 deg C. The time at which the temperature of the solution was held at 95 deg C is shown in Table 1. Endogenous peroxidase was quenched with a 3% solution of hydrogen peroxide in distilled water for 5 minutes. The slides were incubated with the primary antibody at room temperature (Table 1), and immunoreactivity was demonstrated using the LSAB kit (Dako) followed by 3-3'-diaminobenzidine-hydrogen peroxide solution. The slides were counterstained with hematoxylin, dehydrated, and mounted.

For M30 immunostaining, sections were cut, dewaxed, and hydrated as described herein. Then, antigen retrieval was performed in target retrieval solution (Dako) at 98 deg C for 15 minutes. Hydrogen peroxide solution from the EnVision kit (Dako) was applied for 7 minutes, and the slides were washed and incubated with serum-free protein block (Dako). The primary antibody solution, M30 diluted 1:150 in Tris-buffered saline (Dako) with 0.05% Tween 20, was applied for 1 hour at room temperature. After rinsing, labeled EnVision polymer (Dako) was added for 30 minutes, and the presence of bound antibody was demonstrated with the substrate-chromogen solution (hydrogen peroxide and 3-3'-diaminobenzidine) from the EnVision kit. The slides were counterstained with hematoxylin, dehydrated, and mounted.

Morphologic classification of the appendiceal and colorectal lesions conformed to the recommendations of the World Health Organization,30 and staging was according to the TNM system.31 For Ki-67, M30, and p53, the sections were examined at high power; neoplastic cells that exhibited immunoreactivity were expressed as a percentage of the total number of neoplastic cells counted. At least 1000 cells were counted in each case. For bcl-2, a semiquantitative classification was used: the lesion was classified as negative if 5% or less of the cells expressed bcl-2 and as positive if more than 5% of the cells were reactive. This cutoff of 5% has been used by other authors.32 For CD44s, lesions were divided into those that were negative (no immunoreactivity in any cells) and positive (membrane and/or cytoplasm immunoreactivity present).

The data were analyzed using Stat-100 (Biosoft, Cambridge, England) and SPSS statistical software (SPSS Inc, Chicago, Ill) running on Windows NT. A P value of s.05 was considered statistically significant. All chi^sup 2^ tests were performed with Yates continuity correction.

RESULTS

Of the cases originally identified, there was insufficient tissue in the paraffin blocks for analysis in 6, leaving a total of 299 cases suitable for study. One hundred seventeen were patients with adenomas, whereas 182 patients had carcinomas. The demographic data are shown in Table 2, and the staging data for those tumors in which the information was available are shown in Table 3.

In many groups, a few cases had to be excluded because of a failure of internal controls to exhibit a positive reaction or because of insufficient tissue. The numbers of cases included in each group and the results are shown in Table

4. Examples of positive immunoreactions are shown in the Figure.

The Ki-67, M30, and p53 counts showed a skewed distribution and were compared with the Mann-Whitney U test. The bcl-2 and CD44s were compared using chi^sup 2^ tests. The adenomas showed significant differences in Ki-67 (P

Since the ratio between proliferation and apoptosis may be of more relevance than the absolute numbers of proliferating cells and apoptotic cells, the ratio of M30 to Ki-67 was calculated for each case. For adenomas there was no significant difference in the ratio of M30 to Ki-67 between colorectum and appendix, but for carcinomas the median of the ratio was significantly higher in the colon (0.013) than in the appendix (0.003) (P = .01).

Further analysis of the data showed bcl-2 was less likely to be expressed in carcinomas than in adenomas (41 of 165 carcinomas were positive compared with 44 of 110 adenomas; P = .01). CD44s expression was lower in carcinomas (94 of 171) than adenomas (79 of 110, P = .01). We found no correlation between bcl-2 and Ki-67, M30, or p53, nor was there a correlation between p53 and Ki-67 or M30.

There were 34 cases of well-differentiated mucinous carcinomas of the colon and rectum (median age, 62 years) and 33 of the appendix (median age, 52 years). None of the colorectal lesions was associated with pseudomyxoma peritonei, whereas 20 of the appendiceal cases were. Table 5 shows the results. The only variables to show a significant difference between the 2 sites were M30 count (P

COMMENT

Carcinomas displayed more expression of Ki-67, M30, and CD44s in the colorectum than in the appendix, and the ratio of M30 to Ki-67 was also significantly higher. The lower rates of proliferation and apoptosis in the appendiceal carcinomas implied by these findings are in keeping with their more indolent behavior. The decreased expression of CD44s in the appendix might be related to their lower rate of distant metastases, in keeping with the findings of Isozaki et al.33 The lack of any significant difference in bcl-2 and p53 expression makes it unlikely that they could be directly related to differences in behavior.

However, when the analysis was confined to well-differentiated mucinous carcinomas, only M30 expression and the M30:Ki-67 ratio were significantly different. Therefore, the differences in Ki-67 and CD44s may be explained by the different proportions of well-differentiated mucinous carcinomas at the 2 sites, whereas the differences in M30 and the M30:Ki-67 ratio did not depend on the morphologic classification of the tumor. It is, perhaps, surprising that the apoptotic count in the appendiceal mucinous neoplasms should be lower than in the colorectal ones,. given the tendency to slow growth exhibited by the former. The M30:Ki-67 ratio was also lower in the appendix, and this observation does not suggest a concurrent reduction in cell proliferation. A possible explanation is a shorter time taken for apoptosis in mucinous appendiceal tumors, so that apoptotic cells are encountered relatively infrequently. Alternatively, it may be that pseudomyxoma peritonei has a higher apoptotic rate compared with the primary tumors examined in this study.

In our series, adenomas showed significantly greater expression of Ki-67, p53, and bcl-2 in the colorectum than the appendix. It is possible that these differences could be related to the increased proportion of adenomas showing villous or serrated morphologic findings in the appendix. In one study of hyperplastic polyposis, immunohistochemical overexpression of p53 was rarely observed in serrated adenomas and mixed adenomatous-hyperplastic polyps.34

Although the study was not designed to test other relationships among the variables, the data were used to search for associations that had been noted by other authors. Like others,11-14,29 we found that bcl-2 was less likely to be expressed in carcinomas than in adenomas. We also found that CD44s expression was lower in carcinomas than adenomas, as did Neumayer et al.35 Carcinomas showed a positive correlation between counts of Ki-67 and M30 (P = .03; data not shown) as found in many other studies.10,36-39 There was no correlation between bcl-2 and Ki-67, M30, or p53, nor was there a correlation between p53 and Ki-67 or M30, in line with the results of others.10,12,13,15,29,32,37,38,40,41 Some studies have found mucinous carcinomas to show less p53 expression than nonmucinous carcinomas.5,42 In our series, although there was a trend for mucinous carcinomas to have a lower p53 count than nonmucinous carcinomas, this was not statistically significant (P = .09).

Colorectal adenocarcinomas characterized by high levels of microsatellite instability (MSI-H), in contrast to the more common microsatellite stable carcinomas, are usually diploid or near diploid, are usually found in the right colon, are found in younger age groups, are more often mucinous, are more often poorly differentiated, and have an improved prognosis.43-46 Carcinomas of the appendix share some of these features, since they are usually diploid,47 are frequently mucinous,2 and are by definition right-sided. Furthermore, there is evidence that neoplastic progression in some polyps with serrated morphologic features (eg, hyperplastic polyps, serrated adenomas) can occur through a pathway of low-level microsatellite instability (MSI-L).48,49 Many adenomas and hyperplastic lesions of the appendix have serrated morphologic features, and it is interesting to speculate that carcinomas of the appendix might evolve through this MSI-L pathway. Further circumstantial evidence suggesting this possibility is the high level of k-ras mutation in both appendiceal adenomas50 and colorectal MSI-L carcinomas.48 Therefore, some of the differences we noted may be related to the microsatellite instability status of the neoplasms, with a high proportion of appendiceal adenomas and carcinomas showing microsatellite instability. The MSI-L cases have a lower apoptotic count than the MSI-H cases,51 and our study demonstrated a lower apoptotic count in appendiceal tumors compared with colorectal tumors, although any relationship is unlikely to be a simple one. Further studies to test the hypothesis that a high proportion of appendiceal neoplasms follow the "serrated pathway" of microsatellite instability are indicated.

This study was financially supported by the Education and Research Fund of the Royal Hospital Haslar.

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Norman]. Carr, FRCPath; Theresa S. Emory, MD; Leslie H. Sobin, MD

Accepted for publication February 18, 2002.

From the Department of Cellular Pathology, Southampton University Hospitals National Health Service Trust, Southampton, Hampshire, England (Dr Carr), and Department of Hepatic and Gastrointestinal Pathology, Armed Forces Institute of Pathology, Washington, DC (Drs Emory and Sobin).

Reprints: Norman J. Carr, FRCPath, Department of Cellular Pathology, Southampton General Hospital, Mailpoint 2, Tremona Road, Southampton, Hampshire SO16 6YD, England (e-mail: Norman. Carr@suht.swest.nhs.uk).

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